Emily Rumschlag-Booms

Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza

BackgroundAvian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to α2,3-linked SAs, while human influenza A viruses exhibit a preference for α2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.ResultsWe demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both α2,6-linked and α2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.ConclusionOur results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.

Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry

ABSTRACT Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. IMPORTANCE Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.

A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre–messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.

Comparative analysis between a low pathogenic and a high pathogenic influenza H5 hemagglutinin in cell entry.

Avian influenza viruses continue to threaten globally with pandemic potential. The first step in a potential pandemic is the ability of the virus to enter human cells which is mediated by the viral surface glycoprotein hemagglutinin (HA). Viral entry of influenza is dependent upon the processing of the HA0 polypeptide precursor protein into HA1 and HA2 which is mediated by host cellular proteases. The sequence of the cleavage site which is recognized by host proteases has been linked with pathogenesis of various influenza viruses. Here we examined the effects of cleavage site sequences between a highly pathogenic H5N1 strain and a low pathogenic H5N2 strain to determine their effects on viral entry. From this analysis we determined that at the level of viral entry, the only observed difference between the low and high pathogenic strains is their ability to be cleaved by host cellular proteases.

Influenza A Virus Entry: Implications in Virulence and Future Therapeutics

Influenza A viruses have broad host tropism, being able to infect a range of hosts from wild fowl to swine to humans. This broad tropism makes highly pathogenic influenza A strains, such as H5N1, potentially dangerous to humans if they gain the ability to jump from an animal reservoir to humans. How influenza A viruses are able to jump the species barrier is incompletely understood due to the complex genetic nature of the viral surface glycoprotein, hemagglutinin, which mediates entry, combined with the viruss ability to use various receptor linkages. Current therapeutics against influenza A include those that target the uncoating process after entry as well as those that prevent viral budding. While there are therapeutics in development that target entry, currently there are none clinically available. We review here the genetics of influenza A viruses that contribute to entry tropism, how these genetic alterations may contribute to receptor usage and species tropism, as well as how novel therapeutics can be developed that target the major surface glycoprotein, hemagglutinin.

Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa

Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.

A parallel genome-wide RNAi screening strategy to identify host proteins important for entry of Marburg virus and H5N1 influenza virus

BackgroundGenome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening.MethodsTwo genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits.ResultsThe parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study.ConclusionsThis strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.

Abstract A180: Development of a high-throughput screening (HTS) assay to detect splicing inhibitors.

Background and Hypothesis: Alternative splicing (AS) of RNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract binding protein (PTB) is overexpressed in ovarian tumors (He et al, Clin Cancer Res, 10:4652–60, 2004), compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness (He et al, Oncogene 26:4961–8, 2007). PTB is a widely expressed RNA binding protein whose main molecular function is regulating alternative splicing. Because KD of PTB with shRNA impairs ovarian cancer cell growth and other malignant properties, we wished to identify small molecule inhibitors of PTB that will have the same effect as the shRNA. We hypothesized that PTB is a drugable therapeutic target and that its activity in live cells can be monitored by measuring the splicing of a PTB target gene. Accordingly, we have developed a cell-based fluorescent reporter assay for monitoring RNA splicing by using a GFP minigene. This assay allows HTS of small molecule libraries to identify splicing modulator agents that inhibit PTB. Materials and Methods: In this pilot study, we screened 89 NCI-approved oncology drugs (http://.goo.gl/Lf1Jl) in engineered A2780 epithelial ovarian tumor sublines that carried the splicing reporter minigene construct and/or an inducible shRNA against PTB. Three different sublines were used for this assay: (i) a positive control that carries the splicing reporter construct and an inducible shRNA against PTB; (ii) a negative control that carries the splicing reporter construct only; and (iii) a blank control that consist of parental cells only. For the assay, all cells were seeded in 96-well plates (black, clear bottom) at 1.5×106 cells/mL. After 24 h, compounds were added using a PerkinElmer Janus workstation (http://goo.gl/zbK71), and plates were incubated with compounds (final concentration, 1μM) for 48 h at 37C, 5% CO2. Plates were washed inside the sterile workstation with 1× PBS solution before measuring activity. Fluorescence measurements were carried out on a PerkinElmer EnVision plate reader (http://goo.gl/ZxDN8), using filters at 485-nm for excitation and 520-nm for emission. The activation percent was assessed by quantifying fluorescence intensity changes in the compound-treated cells in relation to the positive and negative control cells. Results: We found that our cell-based splicing reporter assay can be use to detect PTB activity in live cells. Moreover, because compounds in this library are intrinsically cytotoxic, 18% (16 of 89) showed toxicity in the A2780 cells. Of importance, this assay was able to detect cytotoxic compounds. These compounds were separated for further investigation to test them at lower concentrations, and to determine whether any of them affect splicing in the reporter assay. Conclusions: This study describes the preliminary assessment of a novel HTS approach to identify small molecule inhibitors of the splicing factor PTB. This cell-based HTS allows large numbers of agents to be tested against engineered cell lines to identify small molecule inhibitors of PTB with the goal of translating positive hits to the clinic. Supported in part by grants RO1 CA40570 and RO1 CA138762 (to WTB), by OCRF (to XH), and by UIC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A180.